Tiron functions as an effective antioxidant alleviating the intracellular reactive oxygen species (ROS) or the acute toxic metal overload. The previous studies have shown that cardiac myocyte apoptosis can be effectively inhibited by tiron administration in streptozotocin (STZ)‐induced diabetic rats, primary neonatal rat cardiomyocytes (NRVMs), and H9c2 embryonic rat cardiomyocytes. However, the underlying signaling mechanism is ill‐defined. In the present study, we found that tiron supplementation significantly inhibited apoptosis of high glucose (HG)‐treated NRVMs and the left ventricular cardiomyocytes from STZ‐diabetic rat, accompanied with a reduction of osteopontin (OPN) levels as well as an inhibition of PKCδ phosphorylation. OPN knockdown protected NRVMs against HG‐induced cell apoptosis. In addition, genetic inhibition of PKCδ mitigated HG‐stimulated enhancement of intracellular OPN levels in NRVMs. These finding indicate that ROS‐mediated activation of PKCδ upregulated OPN expression, leading to cardiac myocyte apoptosis. Interfering with ROS/PKCδ pathway by antioxidants such as tiron provides an optional therapeutic strategy for treatment and prevention of apoptosis‐related cardiovascular diseases including diabetic cardiomyopathy. This article is protected by copyright. All rights reserved.